Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.1 (chymotrypsin)
10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Trypsin digestion of pertussis toxin (PT) preferentially cleaved the S1 subunit at Arg-218 without detectable degradation of the B oligomer. The fragment produced, termed the tryptic S1 fragment, appears to remain associated with the B oligomer. Chymotrypsin digestion of PT also preferentially cleaved the S1 subunit without detectable degradation of the B oligomer. The chymotryptic S1 fragment possessed a slightly lower apparent molecular weight than the tryptic S1 fragment and was more accessible to the respective protease. Trypsin- and chymotrypsin-treated PT and PT required the presence of dithiothreitol and ATP for optimal enzymatic activity. Trypsin-treated PT showed approximately a 2-4-fold higher level of expression of ADP-ribosyltransferase and NAD-glycohydrolase activities than PT. Chymotrypsin-treated PT also exhibited approximately a 2-fold greater level of ADP-ribosyltransferase activity than PT. The observed increase in activity of protease-treated PT was due primarily to a shorter time for activation in PT mediated ADP-ribosylation of transducin. In addition, trypsin-digested PT possessed the same cytotoxic potential for Chinese hamster ovary cell clustering as PT. One possible role for the generation of a proteolytic fragment of the S1 subunit of PT would be to produce a catalytic fragment with increased efficiency for ADP-ribosylation of G proteins in vivo.
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PMID:Protease treatment of pertussis toxin identifies the preferential cleavage of the S1 subunit. 185 Jul 38

This paper presents morphological data on mouse oocyte maturation and fertilization, reviews evidence supporting the existence of a sperm receptor, and suggests future directions for this line of research. We used scanning electron microscopy to examine oocytes under a variety of conditions. The surfaces of mature mouse oocytes are seen to be similar whether maturation occurs in vivo or in vitro. Capacitated sperm (both acrosome-intact and acrosome-reacted) are observed to interact with the microvilli of the oocyte surface. Little is known about oocyte surface proteins that mediate fertilization in mammals. Data of ours and others show that enzyme treatment of live unfertilized eggs interferes with sperm binding. Enzyme treatment (trypsin, chymotrypsin treatment, or pronase) reduces the number of bound sperm, suggesting removal of a surface protein involved in fertilization. Trypsin treatment also causes some lengthening of surface microvilli in a belt surrounding the metaphase II region. After metabolic labeling, proteins of zona-free unfertilized eggs can be identified by SDS-PAGE and autoradiography. Comparison of 1-D gels from untreated and enzyme-treated eggs show the nearly complete disappearance of proteins of 263, 170, 137, 97, and 87 kD after digestion; an increase in a 66 kD protein after trypsin or chymotrypsin; and a major new band of 20 kD after chymotrypsin treatment. Fertilized eggs show the loss of a 255-265 kD band among other changes. Proteins of 97 kD and 87 kD were seen previously by surface labeling (Johnson and Calarco, 1980b), and our 97 kD and 66 kD bands are similar in molecular weight to those identified by Boldt et al. (1989). Taken together, these data identify a few candidate proteins for the role of sperm receptor on the egg surface. Future work should focus on identification of the surface protein(s) which functions physiologically in fertilization by developing fertilization-blocking antibodies. Relatedness to other mammalian sperm receptors and identification of the genes involved would provide valuable information to our understanding of fertilization and to the problems of infertility and contraception.
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PMID:Fertilization of the mouse oocyte. 186 39

Trypsin inhibitor (TI) occurs naturally in many foods from plants, notably soybean protein products. Heat treatment inactivates TI and improves nutritional quality, but residual TI activity of 5 to 20% remains after typical commercial treatments. Chronic feeding of TI or products that contain TI can inhibit trypsin and chymotrypsin, stimulate their secretion, cause hypertrophy and hyperplasia of the pancreas, and lead to adenomas and carcinomas of the exocrine pancreas. In the rat, TI promotes pancreatic carcinogenesis initiated by azaserine. Data needed for possible risk assessment on TI would include 2-year bioassays from animals treated with TI and fed diets carefully controlled for type and amount of fat (which also promotes pancreatic carcinogenesis). The effects of TI on protein nutrition would have to be considered when identifying the maximum tolerated dose. Major reductions in human dietary TI exposure may not be feasible because of the multiple sources of TI, the substantial promotion by other factors such as fat, and the adverse effects of excessive heat on food products. For risk assessment of TI in a particular food, other promotors and the feasibility of decreasing TI intake must be considered.
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PMID:Residue trypsin inhibitor: data needs for risk assessment. 189 96

Signal-transducing G-proteins are heterotrimers composed of GTP-binding alpha subunits in association with a tightly bound complex of beta and gamma subunits. While the alpha subunits are recognized as a family of diverse structures, beta and gamma subunits have also been found as heterogeneous isoforms. To investigate the diversity and tissue specificity of the beta gamma complexes, we have examined homogeneous oligomeric G-proteins from a variety of sources. The beta and gamma subunits isolated from the major-abundance G-proteins from bovine brain, bovine retina, rabbit liver, human placenta, and human platelets were purified and subjected to biochemical and immunological analysis. Protease mapping and immune recognition revealed an identical profile for each of the two distinctly migrating beta isoforms (beta 36 and beta 35) regardless of tissue or G-protein origin. Digestion with V8 protease revealed four distinct, clearly resolved terminal fragments for beta 36 and two for beta 35. Trypsin and chymotrypsin digestion yielded numerous bands, but again each form had a unique profile with no tissue specificity. Tryptic digestion was found to be conformationally specific with the most resistant structure being the native beta gamma complex. With increasing trypsin, the complex was digested but in a pattern distinct from that for denatured beta. In contrast to the two highly homologous beta structures, examination of this set of proteins revealed at least six distinct gamma peptides. Two unique gamma peptides were found in bovine retinal Gt and three gamma peptides in samples of bovine brain derived Go/Gi. Human placental and platelet Gi samples each contained a unique gamma.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:G-protein beta gamma forms: identity of beta and diversity of gamma subunits. 190 14

We determined whether activity of pancreatic enzymes could be maintained in frozen duodenal juice by diluting the specimens or by adding nutrients or a chymotrypsin inhibitor. Human duodenal juice was obtained during cholecystokinin octapeptide IV administration. Trypsin, chymotrypsin, lipolytic, lipase, and colipase activities were measured in fresh undiluted or diluted (1:4 and 1:16 with saline and T-tube bile) duodenal juice as well as after adding CaCl2, casein, triolein, or a chymotrypsin inhibitor. Subsequently, the samples were frozen at -20 degrees C, and enzyme activities were measured at 1, 2, 3, 7, 14, 28, and 56 days. Activities of chymotrypsin and colipase did not change during freezer storage. Trypsin survival was variable in juice from different subjects. By contrast, in duodenal juice to which no nutrient or only CaCl2 had been added, 90%, 65%, and 40% (P = 0.05 vs. undiluted) of lipolytic activity was lost by 56 days in undiluted and 1:4 or 1:16 diluted duodenal juice samples, respectively. The loss of lipolytic activity was prevented (P less than 0.05) by adding casein or casein and triolein to undiluted and 1:4 diluted samples and turkey egg white to undiluted samples. The loss of lipolytic activity was strongly associated with loss of lipase activity (r = 0.97) but only weakly associated with loss of colipase activity (r = 0.49). In summary, chymotrypsin and colipase are well preserved in frozen duodenal juice and can be used to accurately assess concentrations of pancreatic enzymes after thawing frozen duodenal samples. If it is necessary to measure lipolytic activity after freezing samples, lipase can be maintained by adding casein or a chymotrypsin inhibitor to juice before freezing.
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PMID:How to protect human pancreatic enzyme activities in frozen duodenal juice. 198 21

Trypsin inhibitory activity from the hemolymph of Limulus polyphemus was found to co-purify with coagulogen (the clottable protein in blood coagulation) after acidification, ammonium sulfate precipitation, and gel filtration. Limulus trypsin inhibitor (LTI) was separated from coagulogen by ion-exchange chromatography on carboxymethyl-Sephadex. LTI is an inhibitor of trypsin (Ki = 3.3 nM) on both high and low molecular weight substrates. It also inhibits chymotrypsin but has little or no effect on thrombin, thermolysin, pepsin, or papain, nor does LTI inhibit the proteolytic cascade produced in endotoxin-stimulated Limulus amoebocyte lysate coagulation. Electrophoresis under nonreducing conditions on denaturing polyacrylamide gel yields a doublet migrating with an estimated Mr of 20,000. Under reducing conditions, a single broad band migrates with an estimated Mr of 15,000. The native structure is a monomer of moderate asymmetry with a molecular weight of 16,300 and a so20,w = 1.5(5), as determined by analytical ultracentrifugation. The amino acid composition of LTI yields a calculated molecular weight of 15,680 and a calculated partial specific volume of 0.71(7) ml/g. LTI does not contain methionine, tryptophan, or detectable levels of reducing carbohydrate. The NH2-terminal sequence (V-S-P-P-F-I-K-Q-T-K-F-S-T-X-F-L-G-X-S-S) consists primarily of hydrophobic amino acid residues. Comparison of the amino acid composition and amino-terminal sequence of LTI with those of other known protease inhibitors reveals no significant similarity to other trypsin inhibitors. The novel physical characteristics suggest that LTI represents a new type of protease inhibitor.
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PMID:A novel trypsin inhibitor from the hemolymph of the horseshoe crab Limulus polyphemus. 198 74

The mitochondrial energy-linked nicotinamide nucleotide transhydrogenase is a homodimer of monomer Mr = 109,228. Hydropathy analysis of its cDNA-deduced amino acid sequence (1043 residues) has indicated that the molecule is composed of 3 domains: a 430-residue-long hydrophilic N-terminal domain which binds NAD(H), a 200-residue-long hydrophilic C-terminal domain which binds NADP(H), and a 400-residue-long hydrophobic central domain which appears to be made up mainly of about 14 hydrophobic clusters of approximately 20 residues each. In this study, antibodies were raised to the hydrophilic N- and C-terminal domains cleaved from the isolated transhydrogenase by proteolytic digestion, and to a synthetic, hydrophilic pentadecapeptide, which corresponded to position 540-554 within the central hydrophobic domain. Immunochemical experiments with mitoplasts (mitochondria denuded of outer membrane) and submitochondrial particles (inside-out inner membrane vesicles) as sources of antigens showed that essentially the entire N- and C-terminal hydrophilic domains of the transhydrogenase, as well as epitopes from the central pentadecapeptide, protrude from the inner membrane into the mitochondrial matrix, where the N- and C-terminal domains would be expected to come together to form the enzyme's catalytic site. Treatment of mitoplasts with several proteolytic enzymes indicated that large protease-sensitive masses of the transhydrogenase are not exposed on the cytosolic side of the inner membrane, which agreed with the exception that the central highly hydrophobic domain of the molecule should be largely membrane-intercalated. Trypsin, alpha-chymotrypsin, and papain had little or no effect on the mitoplast-embedded transhydrogenase. Proteinase K, subtilisin (Nagarse), thermolysin, and pronase E each split the mitoplast-embedded enzyme into two fragments only, a fragment of approximately 70 kDa containing the N-terminal hydrophilic domain, and one of approximately 40 kDa bearing the C-terminal hydrophilic domain. The cleavage site of proteinase K was determined to be A690 -A691, which is located in a small hydrophilic segment within the central hydrophobic domain. This protease-sensitive loop appears to be exposed on the cytosolic side of the inner membrane. The proteinase K-nicked enzyme containing two peptides of 71 and 39 kDa was isolated from mitoplasts and shown to have high transhydrogenase activity.
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PMID:Mitochondrial energy-linked nicotinamide nucleotide transhydrogenase. Membrane topography of the bovine enzyme. 200 10

Alanyl-tRNA synthetase from Escherichia coli, Bombyx mori and rat were examined with respect to the following functional and structural properties: the effect of substrates on sensitivity to proteolysis, secondary structure as determined by circular dichroism, amino acid composition and, in the case of the rat and insect enzymes, partial amino acid sequence determination on a 60-kDa C-terminal tryptic fragment. Digestion of the enzyme from all three sources with trypsin resulted in significant decline in aminoacyl-tRNA synthetase activity with little effect on pyrophosphate-exchange activity. In each case the presence of alanine and ATP together, but not separately, reduced the rate of digestion by trypsin; the largest effect was observed with the enzyme from rat liver. Trypsin digestion generated fragments of 47 kDa and 40 kDa with all three enzymes, but detection of significant quantities of the 47-kDa fragment from the rat enzyme required the presence of ATP and alanine. Trypsin digestion produced a fragment of 60 kDa with all three enzymes, but detection of significant quantities of this fragment with the bacterial enzyme required the presence of ATP and alanine. Limited sequence analysis of the 60-kDa fragment from the insect and rat enzymes indicated that trypsin cleaved both proteins at the same site to generate this species. Similar effects of substrates were observed when the enzymes were digested with chymotrypsin suggesting that the effects of substrates on protease sensitivity were not unique to trypsin. Circular dichroism spectra obtained for the three enzymes were qualitatively and quantitatively similar. There is some similarity in amino acid composition between the rat and insect enzymes.
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PMID:Alanyl-tRNA synthetase from Escherichia coli, Bombyx mori and Ratus ratus. Existence of common structural features. 204 Feb 80

Protease nexin-2 (PN-2) is a protease inhibitor that is synthesized and secreted by a variety of extravascular cells including human fibroblasts. It forms sodium dodecyl sulfate-stable complexes with trypsin, the epidermal growth factor binding protein and the gamma-subunit of nerve growth factor. Recently we reported that PN-2 is the secreted form of the amyloid beta-protein precursor (APP) and is a potent inhibitor of chymotrypsin. Here we describe a two-step procedure to purify PN-2/APP using a monoclonal antibody immunoaffinity column. We also quantitated the protease inhibitory properties of purified PN-2/APP on a number of serine proteases. PN-2/APP was a potent inhibitor of coagulation factor XIa with a Ki = 2.9 x 10(-10). The inhibition of factor XIa by PN-2/APP was augmented by heparin and resulted in a Ki = 5.5 x 10(-11) M. Trypsin and chymotrypsin were also effectively inhibited with a Ki = 4.2 x 10(-10) and 1.6 x 10(-9), respectively. PN-2/APP also inhibited the epidermal growth factor binding protein, the gamma-subunit of nerve growth factor, and chymase and plasmin to a lesser extent. In view of recent findings that PN-2/APP is contained in alpha-granules of platelets and is secreted upon platelet activation, the potent inhibition of factor XIa suggests that PN-2/APP may play a regulatory role in the coagulation pathway at vascular wound sites. In addition, these studies define biochemical activities of PN-2/APP which may be involved in regulating proteases that lead to the generation and deposition of the beta-protein in neurodegenerative lesions associated with Alzheimer's disease and Down's syndrome.
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PMID:Immunopurification and protease inhibitory properties of protease nexin-2/amyloid beta-protein precursor. 211 43

Procollagenase of human polymorphonuclear leucocytes was purified to homogeneity using a rapid and reproducible method. The purification procedure included affinity chromatography on zinc chelate Sepharose, ion exchange chromatography on Q-Sepharose fast flow, followed by affinity chromatography on orange Sepharose and finally a gel-permeation step on Sephacryl S-300. It was shown by SDS/PAGE, under reducing conditions, that the latent collagenase of human polymorphonuclear leucocytes consists of a single polypeptide chain with an apparent relative molecular mass of 85,000. Upon deglycosylation by endoglycosidase F digestion, the apparent relative molecular mass of the procollagenase was reduced to 53,000 which is similar to that of the fibroblast enzyme, and indicates a close relationship between both enzymes. Sequence data were determined by direct automated Edman degradation of the purified polymorphonuclear leucocyte procollagenase. The complete sequence of the propeptide region (residue 1-120) was thereby established. The proteolytic activation of the polymorphonuclear leucocyte procollagenase by various enzymes was investigated by determining the N-terminal sequences of the intermediate and final activated forms. Activation by chymotrypsin and cathepsin G led to the active form (Mr 64,000) by cleaving 79 N-terminal residues from the proenzyme. Trypsin activates in a two-step process. Cleavage of 48 N-terminal residues led to a still latent Mr 70,000 species. The final active form (Mr 65,000) was obtained by splitting off 20 additional N-terminal residues.
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PMID:Characterization and activation of procollagenase from human polymorphonuclear leucocytes. N-terminal sequence determination of the proenzyme and various proteolytically activated forms. 215 79


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